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Unveiling Genomic Regions That Underlie Differences Between Afec-Assaf Sheep and Its Parental Awassi Breed

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Unveiling Genomic Regions That Underlie Differences Between Afec-Assaf Sheep and Its Parental Awassi Breed Seroussi et al. Genet Sel Evol (2017) 49:19 DOI 10.1186/s12711-017-0296-3 Genetics Selection Evolution RESEARCH Open Access Unveiling genomic regions that underlie differences between Afec‑Assaf sheep and its parental Awassi breed Eyal Seroussi, Alexander Rosov, Andrey Shirak, Alon Lam and Elisha Gootwine* Abstract Background: Sheep production in Israel has improved by crossing the fat-tailed local Awassi breed with the East Friesian and later, with the Booroola Merino breed, which led to the formation of the highly prolific Afec-Assaf strain. This strain differs from its parental Awassi breed in morphological traits such as tail and horn size, coat pigmentation and wool characteristics, as well as in production, reproductive and health traits. To identify major genes associated with the formation of the Afec-Assaf strain, we genotyped 41 Awassi and 141 Afec-Assaf sheep using the Illumina Ovine SNP50 BeadChip array, and analyzed the results with PLINK and EMMAX software. The detected variable genomic regions that differed between Awassi and Afec-Assaf sheep (variable genomic regions; VGR) were compared to selection signatures that were reported in 48 published genome-wide association studies in sheep. Because the Afec-Assaf strain, but not the Awassi breed, carries the Booroola mutation, association analysis of BMPR1B used as the test gene was performed to evaluate the ability of this study to identify a VGR that includes such a major gene. Results: Of the 20 detected VGR, 12 were novel to this study. A ~7-Mb VGR was identified on Ovies aries chromo- some OAR6 where the Booroola mutation is located. Similar to other studies, the most significant VGR was detected on OAR10, in a region that contains candidate genes affecting horn type (RXFP2), climate adaptation (ALOX5AP), fiber diameter (KATNAl1), coat pigmentation (FRY) and genes associated with fat distribution. The VGR on OAR2 included BNC2, which is also involved in controlling coat pigmentation in sheep. Six other VGR contained genes that were shown to be involved in coat pigmentation by analyzing their mammalian orthologues. Genes associated with fat distribution in humans, including GRB14 and COBLL1, were located in additional VGR. Sequencing DNA from Awassi and Afec-Assaf individuals revealed non-synonymous mutations in some of these candidate genes. Conclusions: Our results highlight VGR that differentiate the Awassi breed from the Afec-Assaf strain, some of which may include genes that confer an advantage to Afec-Assaf and Assaf over Awassi sheep with respect to intensive sheep production under Mediterranean conditions. Background Awassi dairy strain [2]. The Assaf dairy breed was formed In Israel, within-breed selection, crossbreeding and gene about 30 years later by crossing the improved Awassi introgression have contributed to the transition of the with the European East Friesian (EF) breed [3]. In 1986, sheep industry from traditional extensive production breeding of the highly prolific Afec-Assaf strain was ini- using the native fat-tailed local Awassi breed to highly tiated by introgressing the Booroola mutation (B) at the intensive production with the Assaf and Afec-Assaf BMPR1B gene into the Assaf breed by crossing Assaf sheep [1]. Selection within the local Awassi sheep that ewes with Booroola Merino rams [4–6]. The high prolifi- began in the 1930s led to the formation of the improved cacy of the Afec-Assaf strain is due to the presence of the B allele, which is inherited in an almost completely domi- nant mode [7], since BB and B ewes have similar pro- *Correspondence: [email protected] + Institute of Animal Science, ARO, The Volcani Center, PO Box 15159, lificacy. However, since the perinatal lamb survival rate is 7528809 Rishon LeZion, Israel higher in B+ than in BB ewes, B+ is the recommended © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 2 of 10 genotype for females in commercial flocks [7]. The B By using the ovine single nucleotide polymorphism allele segregates in the Afec-Assaf population, thus gen- (SNP) 50 BeadChip array (Illumina Inc., San Diego, CA) otyping lambs for the Booroola mutation and selecting and BMPR1B as a test gene, the aims of our study were only the B+ ewe lambs as replacements is a common to: (1) compare the Awassi and Afec-Assaf genomes, practice in Afec-Assaf flocks to maintain high prolificacy searching for variable genomic regions (VGR) that differ in the long term. Afec-Assaf sheep differ widely from the between the two breeds; and (2) link these VGR to genes Awassi sheep in a number of phenotypic characteristics, and selection signatures that were previously described such as the presence of horns, tail shape, coat color pig- in sheep GWAS. mentation and wool quality, as well as seasonality, prolifi- cacy, and milk production [8, 9] (Table 1). Methods In sheep, genome-wide association studies (GWAS) Ethics statement have contributed to better understanding of their domes- Experimental protocols were approved (Approval No. IL tication [10, 11], as well as to the identification of specific 415-12) by the Volcani Center Institutional Animal Care genomic regions that are associated with differences in and Use Committee. various traits between breeds (see Additional file 1: Table S1). GWAS have made it possible to identify candidate Animals genes that underlie various phenotypic differences, for Local Awassi sheep are raised by Bedouin growers in instance in Texel sheep RXFP2 for horn type [12] and small unconnected flocks that are maintained under disease-resistance genes such as PITX3 and DMP1 which extensive traditional management with no records. The are involved in microphthalmia [13] and in Corriedale Awassi cohort consisted of 24 local Awassi rams that sheep for rickets [14]. included all of the rams from five flocks, and 17 improved Cryptic relatedness, which occurs when there is Awassi ewes from the Ein-Harod flock. Since the degree unknown kinship within the sample, and population of relationship between animals from the same flock stratification due to random genetic drift in the sam- is unknown, the effective number of local Awassi indi- ple’s subpopulations are two major confounding effects viduals might be smaller than sampled. However, this that cause spurious associations in GWAS analyses [15]. was not the case, since a genomic relationship analysis Hence, incorporating a known genetic marker that is (see Additional file 2: Table S2) showed no strong kin- located within a major gene that segregates within the ship between individuals within or among flocks. The tested population into the GWAS analysis may assist Afec-Assaf cohort consisted of 22 males and 119 females in verifying the power of the study for detecting selec- from the experimental flock of the Volcani Center at Bet tion signatures. This has been the case in several sheep Dagan and two commercial farms. Genotyping indicated GWAS, in which the MC1R gene that affects coat pig- that 35, 95 and 11 animals of the Afec-Assaf cohort were mentation [16] was used as the ‘test’ gene, and poly- homozygous BB, heterozygous B+ or non-carrier ++ for morphism at the GDF8 gene associated with muscle the Booroola mutation, respectively. As expected [7], the hypertrophy [17] was used to establish criteria to detect average prolificacy of the BB and B+ females (based on 3 selection signatures. to 9 parity records per animal) was high, i.e. 3.1 and 3.0 Table 1 Phenotypic, production and reproductive traits of local Awassi, improved Awassi, Assaf and Afec-Assaf sheep Traits Local Awassi Improved Awassi Assaf Afec-Assaf Body size Smalla Largea Largea Largea Tail shape Fat tail Fat tail Partial fat tail Partial fat tail Horn shape Most rams and ewes are Most males and females are polled, horned or carry scurs but some carry short knobs or scurs Wool type Coarse Coarse Medium-coarse Medium-coarse Coat pigmentation Most sheep have brown Most sheep have white face face and white fleece and white fleece Seasonality Seasonal Seasonal Moderately seasonal Moderately seasonal Adaptation to Middle-Eastern High High Intermediate Intermediate climate Milk production Low High High High Prolificacy (lambs born/ewe 1.2 1.3 1.6 2.5 lambing) a Small: ram body weight ~70 kg; large: ram body weight ~110 kg Seroussi et al. Genet Sel Evol (2017) 49:19 Page 3 of 10 lambs born/lambing, respectively. No lambing records Identification of candidate polymorphisms were available for non-carrier ++ females. Exon sequences of the candidate genes were downloaded from NCBI (https://www.ncbi.nlm.nih.gov) and genomic Genotyping using the ovine 50 K beadchip reads were mapped to these templates using GAP5 soft- DNA was extracted from blood samples using the ware [20]. BWA options for this mapping were set to bam DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) bwasw -t 8 -T 30 [21]. Genetic markers were identified according to the manufacturer’s instructions. DNA sam- and called by comparing the consensus contig sequences ples were genotyped by the Ovine SNP50 BeadChip of the Awassi and Assaf individuals with the GAP5 con- assay using standard procedures (http://www.illumina. tig comparator and the heterozygosity search option com). All the Awassi and some of the Afec-Assaf sam- (value = 10). ples were genotyped as part of the International Sheep Genomic Consortium initiative (http://www.sheephap- Scoring amino acid substitutions map.org).
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